|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
|
Fig. 7. Differential stability of cell-surface-associated and secreted RGMc proteins in Cos-7 cells. (A) Time course of the decrease in RGMc protein expression in whole-cell protein extracts (WCE) and conditioned culture medium (CM) from Cos-7 cells infected 12 hours earlier with Ad-HA-RGMc and treated with cycloheximide and doxycycline for the times indicated. Immunoblotting for cadherin demonstrates effective inhibition of new cellular protein synthesis and immunoblotting for tubulin and albumin staining show equivalent sample loading (lower panels). Graphical analysis of average band intensity from at least two independent experiments is displayed to the right. (B) Time course of decline in cell-surface RGMc after inhibition of protein synthesis with cycloheximide. Membrane-associated RGMc was labeled by treating cells infected 12 hours earlier with Ad-HA-RGMc with the EZ-link reagent for 30 minutes, followed by addition of cycloheximide and doxycycline for up to 24 hours. Biotin-labeled RGMc or cadherin was detected by immunoblotting following streptavidin-agarose pull-down from WCE. An immunoblot for tubulin (lower panels) is included as a loading control. Average cell-surface expression for two independent experiments of full-length RGMc (fl), RGMc N- and C- terminal fragments (N/C), and cadherin proteins is shown in the graph to the right. (C) Time course of accumulation of biotin-labeled RGMc in conditioned culture medium following treatment of cells with the EZ-link reagent and incubation with cycloheximide and doxycycline for the times indicated. Total and biotin-labeled RGMc in CM was determined by immunoblotting as outlined in B. Average levels for two independent experiments of total and biotin-labeled RGMc are plotted to the right. Black and gray arrows in blots indicate full-length and internally cleaved RGMc proteins, respectively.
|
